阅读说明：本技术 一种髓细胞触发受体2的激活用药及其应用 (Medicine for activating marrow cell trigger receptor 2 and application thereof ) 是由 孙秀兰 薛腾飞 程虹 郭若冰 杨进 季娟 范益 胡刚 于 2019-11-06 设计创作，主要内容包括：本发明公开了磷酸化芬戈莫德在制备髓细胞触发受体2的激活用药中的应用,应用于调节细胞吞噬。本发明首次披露了磷酸化芬戈莫德是TREM2的激动剂,并揭示了磷酸化芬戈莫德通过激活TREM2增强小胶质细胞的吞噬功能,阐明了磷酸化芬戈莫德调节小胶质细胞吞噬功能的新机制。(The invention discloses application of phosphorylated fingolimod in preparation of a medicine for activating a myeloid cell trigger receptor 2, and the phosphorylated fingolimod is applied to regulation of phagocytosis of cells. The invention discloses that phosphorylated fingolimod is an agonist of TREM2 for the first time, and discloses that phosphorylated fingolimod enhances the phagocytic function of microglia by activating TREM2, and a novel mechanism for regulating the phagocytic function of the microglia by phosphorylated fingolimod is elucidated.)

1. The application of phosphorylated fingolimod in preparing a medicament for activating a myeloid cell trigger receptor 2 is characterized by being applied to regulating phagocytosis of cells.

2. Use of phosphorylated fingolimod in the manufacture of a medicament for the activation of myeloid cell triggering receptor 2 according to claim 1 for enhancing the phagocytic function of microglia.

3. Use of phosphorylated fingolimod in the manufacture of a medicament for activation of myeloid cell triggering receptor 2 according to claim 2 wherein phosphorylated fingolimod enhances microglial phagocytic function by activating myeloid cell triggering receptor 2.

4. Use of phosphorylated fingolimod in the manufacture of a medicament for the activation of myeloid cell triggering receptor 2 according to claim 1 for the modulation of inflammation.

Technical Field

The invention relates to the technical field of drug research and development, in particular to an activating drug for a marrow cell trigger receptor 2 and application thereof.

Background

Microglia are innate immune effector cells in the central nervous system. In the normal development of a nervous system, microglia plays the functions of rapidly clearing apoptotic neurons, trimming synapses of the neurons, secreting cytokines and the like to regulate the survival of the neurons and the like; in pathological conditions, microglia is one of the earliest responding immune cells. For example, in cerebral injury such as cerebral arterial thrombosis, along with the death of a large number of neurons, the dead neurons release various dangerous molecules such as nucleic acid, protein and lipid, and further cause inflammatory reaction, and if the cytotoxic cell fragments cannot be effectively removed, the plasticity of the injured neurons can be weakened, secondary inflammation is initiated, and injury is aggravated. Therefore, modulation of microglial phagocytosis is one of the important pathways for alleviating nerve damage.

The myeloid cell-triggered receptor 2(TREM2) is a type I single transmembrane protein, a member of the immunoglobulin receptor family. TREM2 is highly expressed on macrophages, dendritic cells, osteoclasts, and microglia. Within the central nervous system, TREM2 is specifically expressed on microglia. Because of the lack of intracellular segment, TREM2 should realize signal transduction through its co-receptor 12kDa DNAX activating protein (DAP12), and finally play the roles of regulating phagocytosis, promoting cell growth, inhibiting apoptosis, regulating inflammation, etc. To date, while numerous agents have been found to bind to TREM2 to trigger downstream signals, such as phospholipids, nucleic acids, proteoglycans, heat shock proteins 60 and apolipoproteins, no agents that activate TREM2 have been found.

Phosphorylated fingolimod (FTY 720-phospate, FTY720-P) is an active form of fingolimod (FTY720) formed in vivo under the catalysis of sphingosine kinase 2(SphK2), is a structural analogue of sphingosine-1-Phosphate (S1P), can activate S1PR1/3/4/5, and is involved in many physiological functions mediated by it, such as cell survival, apoptosis, proliferation, and the like. The inventor newly finds that FTY720 has the function of enhancing the phagocytosis of microglia, but whether the phagocytosis promoting function is mediated by activating a phagocytosis related receptor of the microglia, such as a marrow cell trigger receptor 2(TREM2) is not reported.

Disclosure of Invention

The invention aims to provide application of phosphorylated fingolimod in preparation of a medicine for activating a myeloid cell trigger receptor 2, so as to solve the problems in the prior art, and determine that phosphorylated fingolimod is an activator of the myeloid cell trigger receptor 2 by determining the relationship between phosphorylated fingolimod and the myeloid cell trigger receptor 2.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of phosphorylated fingolimod in preparation of a medicine for activating a myeloid cell trigger receptor 2, and the phosphorylated fingolimod is applied to regulation of phagocytosis of cells. The structural formula of the phosphorylated fingolimod (FTY720-P) is shown as a formula I,

preferably, the application is to enhance the phagocytic function of microglia.

Preferably, the phosphorylated fingolimod enhances the phagocytic function of microglia by activating myeloid cell trigger receptor 2.

Preferably, the application is in the regulation of inflammation. Specifically, the method comprises the following steps: phosphorylated fingolimod modulates inflammation by activating S1PR 1/3/4/5.

The invention discloses the following technical effects:

the present invention uses computer modeling to predict the likelihood of binding of human derived TREM2(hTREM2) and FTY720-P, showing that hTREM2 binds to FTY720P via arginine 47, serine 65 and arginine 77 (as shown in figure 1). FTY720-P was further demonstrated to bind directly to TREM2 by a Microcalorimetric Surge (MST) method, indicating that FTY720-P is a ligand of TREM 2. In addition, during the coculture of neurons and microglia, after oxygen deprivation-reperfusion injury, the FTY720 treatment can enhance the phagocytosis of the microglia on the neuron fragments, and after TREM2 is knocked down to express, the phagocytosis promoting function of the FTY720 is obviously weakened, and further, the function of FTY720-P in promoting phagocytosis by activating TREM2 receptors is shown from the functional perspective. The invention discloses that FTY720-P is an agonist of TREM2, the FTY720-P can enhance the phagocytic function of microglia by acting on TREM2, promote the removal of cell debris under the condition of pathological injury, and disclose a new function and a new mechanism of FTY720 for regulating the function of the microglia.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a graph showing the molecular docking results of TREM2 according to the present invention;

FIG. 2 is a graph showing the results of Microcalorimetric Surge (MST) in example 1 of the present invention;

FIG. 3 is a diagram showing the results of immunofluorescence staining of microglia and neurons in co-labeling according to the present invention; wherein, a: WB validation of knockdown efficiency of TREM2 RNA interference (RNAi) on microglia; b: carrying out double immunofluorescence staining on the cells by Iba1 and NeuN to reflect the phagocytosis condition of microglia; c: counting the proportion of phagocytized microglia;

FIG. 4 is a graph showing the results of immunofluorescence co-labeling of Iba1 with CD68 in accordance with the present invention; wherein, a: the results of immunofluorescence staining with Iba1 and CD 68; b: counting the expression level of CD68 based on fluorescent staining;

FIG. 5 is a graph showing the binding relationship between hTREM2 and FTY720 detected by the Microcalorimetric Surging (MST) method of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.